ABSTRACT: PROJECT 2 Although circulating high density lipoproteins (HDL) are considered protective from cardiovascular disease, we have a remarkably limited understanding of their structure. Furthermore, we understand even less about how the major HDL protein, apolipoprotein (apo)A-I, interacts with other proteins to dictate HDL function. We will test the hypothesis that apoA-I makes highly specific contacts with itself to form a molecular scaffold that stabilizes HDL and facilitates, through specific protein:protein interactions, the association of HDL partner proteins to define particle function. In our previous work, we used cross-linking chemistry and mass spectrometry to generate detailed models of apoA-I in reconstituted particles as well as ?real? HDL from human plasma. Despite substantial differences in size and shape, these structures all shared the theme of an antiparallel belt-like arrangement. Building on these discoveries, our goal is to further evaluate these and other models using complementary structural techniques as well as evaluate the basis of apoA-I's interactions with three major HDL components: apolipoprotein A-II, paraoxonase 1 (PON1) and cholesteryl ester transfer protein (CETP). The specific aims are: 1) To test the Trefoil and other models of apoA-I in spherical reconstituted and native or ?real? plasma HDL using new dual isotope cross-linking techniques and state-of-the-art all-atom and course grained molecular dynamics (MD) techniques in synergy with Segrest and Core B. 2) To determine the molecular interactions between apoA-I and apoA-II using cross-linking and a new human apoA-II bacterial expression system to derive the first models of native HDL particles containing both proteins (also in synergy with Segrest). 3) To determine the molecular interactions between apoA-I and two important HDL docking proteins, PON1 and CETP, using chemical cross-linking and site-directed mutagenesis. Along the way, we will also use our experimental techniques to directly test structural models of LCAT being generated by Segrest in Project 1 and evaluate the structure of potentially enhanced functional HDL isolated from lecithin:cholesterol acyl transferase deficient subjects studied by Heinecke in Project 3. Our approach uniquely intertwines new experimental techniques with state-of-the-art MD approaches resulting in structural knowledge that will be directly applied to HDL function. Furthermore, our focus is on the structure of authentic HDL particles that are circulating in normal individuals as well as those with rare genetic disorders. The structure of apoA-I undoubtedly modulates HDL metabolism, and possibly mediates cardioprotective effects of some HDL subspecies. Thus, a molecular understanding of its structure and its interactions with other proteins, particularly those being explored as drug targets such as LCAT and CETP, is critical for the design of new therapies exploiting reverse cholesterol transport and the anti-inflammatory roles of HDL.